Project Summary/Abstract The overall goal of our work is to understand the assembly and regulation of motile cilia. Cilia play vital motile and signaling roles in virtually every differentiated cell. As a consequence, failure in conserved assembly mechanisms, including the transport machine IFT (intraflagellar transport) and assembly of dynein motors, results in a wide range of human diseases including primary cilia dyskinesia (PCD) and consequences for development and adult organ function. Although we have a good understanding of ciliary structure and dynein motor organization, we are only beginning to understand how the dynein motor complexes are transported and assembled. Gaps in knowledge include steps and factors required for cytoplasmic ?preassembly? of axonemal dyneins, transport into the ciliary compartment, loading of complexes on IFT and regulation of transport, regulation of cargo-IFT unloading and targeting and docking of complexes on outer doublet microtubules in a 96nm repeat organization. The current focus of our work is on assembly of the ciliary axonemal inner dynein arm called I1 dynein, also known as dynein f, as an ideal model for axonemal dynein transport and assembly in the axoneme. I1 dynein is a large, conserved, two-headed inner dynein arm that is required for normal ciliary movement and regulation of axonemal bending. I1 dynein is a ?slow?, possibly non-canonical, axonemal dynein thought to resist microtubule sliding driven by other ?fast? axonemal dyneins. As such, I1 dynein, plays a critical role in control of axonemal bending and waveform. We have also determined that I1 dynein is preassembled in the cytoplasm, transported by IFT and targeted to a unique position repeating every 96 nm along each of the doublet microtubule, and assembles independent of the assembly of the other dyneins. Here we will address the transport of I1 dynein, as a cargo for IFT, and test the hypothesis that the Chlamydomonas IDA3 gene, which encodes a coiled-coil protein, Ida3, is specifically required for IFT transport of I1 dynein (Aim 1). We also test the hypothesis that the intermediate chain IC140 interacts with a docking protein or proteins for localization of I1 on the doublet microtubule (Aim 2). From this new work, we will learn how dyneins, as cargoes, are transported to and within cilia, and address the principle that each axonemal complex (e.g. dyneins, radial spokes, N-DRC) require specialized adaptors or regulators for directed transport. In addition, we will, for the first time, learn how an axonemal inner dynein arm is localized in the 96 nm repeat.